It has long been known that the left and right hemispheres of the human brain are anatomically and functionally different, yet how cortical asymmetries are generated, or perturbed in neurological disorders, is poorly understood. In contrast, significant progress has been made towards unraveling the molecular genetic basis for asymmetric development of visceral organs. Components of the Nodal signaling pathway, that function in regulating visceral asymmetry, are also specifically expressed on the left side of the developing zebrafish brain. This transient, left-sided gene expression localizes to the pineal organ anlage. Fish lacking asymmetric gene expression later show a randomization in the left-right positioning of the stalk of the mature pineal gland. Other asymmetries in the diencephalon of lower vertebrates include the left-sided parapineal organ and morphological differences between the left and fight habenular nuclei. The overall goal of this proposal is to determine how diencephalic asymmetries arise in the zebra fish brain. In AIM I, pineal development will be monitored real-time using transgenic zebrafish. Preliminary data suggest that the parapineal is derived from the pineal anlage. Fate mapping will address the cellular origin of the parapineal directly. The parapineal appears to influence transcription in the adjacent dorsal habenula, creating a left-right difference in habenular gene expression. In AIM II, a collaboratory project will lead to the discovery of new molecular asymmetries in the larval diencephalon. The hypothesis that the parapineal regulates the laterality of the habenulae will be tested by examining expression of habenular markers in mutants with altered left-right patterning, or following selective ablation of the parapineal. In AIM III, a focused genetic screen will identify new ENU-induced mutations that affect the formation of diencephalic asymmetries. Two mutations isolated in a pilot screen that lose habenular asymmetry will be characterized further in AIM IV, Together, the proposed experiments will provide a greater understanding of the genetic mechanisms that underlie left-right specialization of the vertebrate forebrain.